The umbrella cells lining the mucosal surface of the urinary bladder form a barrier between the urine and the underlying muscle layers and vasculature. The barrier function of the uroepithelium depends on several factors including: (i) formation of a polarized epithelium with tight junctions; (ii) presence of a surface glycan layer that prevents bacterial adhesion and may also help prevent passage of urine components across the epithelium; (iii) presence of an apical membrane highly impermeable to water, urea, and toxic components of urine; and (iv) the ability of the barrier to accommodate and respond to large changes in bladder volume. Although the umbrella cell barrier is disrupted in several diseases including interstitial and bacterial cystitis, after spinal cord injury, and upon outlet obstruction, little is understood about how umbrella cells modulate the components of their apical membrane, or how they respond to changes in hydrostatic pressure as the bladder fills and empties. The overall hypothesis to be tested in this grant is that umbrella cells respond to changes in hydrostatic pressure by the regulated insertion and/or retrieval of membrane at the apical plasma membrane of the umbrella cells. In Aim I pulse-chase analysis coupled with vesicle and plasma membrane isolation will be used to determine whether discoidal vesicles fuse with the apical plasma membrane in response to hydrostatic pressure. Hydrostatic pressure also stimulates endocytosis. The mechanism of this internalization pathway and how it is regulated will be further characterized. In Aim II the molecular basis of hydrostatic pressure-induced exocytosis will be explored. It is hypothesized that Rab27/myosin Va, Ca z+, and synaptotagmin VII play a role in this process. We will analyze discoidal vesicle exocytosis in mice that lack expression of two isoforms of Rab27 (Rab27a and Rab27b) and myosin Va. The upstream events that stimulate rises in cytoplasmic Ca 2+ will be defined as will the role of synaptotagmin VII, which is one member of a family of proteins that link increased cytoplasmic Ca2v to vesicle fusion. Experiments in Aim III will examine the hypothesis that membrane that was added to the apical surface of umbrella cells during filling will be endocytosed and degraded while discoidal vesicles are replenished by de novo synthesis. Endocytosis will be measured in model systems that mimic bladder filling and voiding. Labeling of internalized surface membrane and inhibitors of vesicle synthesis will be used to determine if post-voiding replenishment of discoidal vesicles is the result of endocytosis or new synthesis.